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April 21, 2006 

CENTER FOR VETERINARY BIOLOGICS NOTICE NO. 06-11 

Subject: Replacement of Potency Assay Reference Samples for Immunodiagnostic 

 Test Kits 

To: Biologics Licensees, Permittees, and Applicants 

 Veterinary Services Management Team 

 Directors, Center for Veterinary Biologics 

 Area Veterinarians in Charge, VS 

 State Veterinarians 

Veterinary Services Memorandum (VS Memo) 800.73, General Requirements for 

Immunodiagnostic Test Kits for the Detection of Antibody or Antigen, provides guidance 

to licensees, permittees, and applicants concerning the requirements to support an 

application for a U.S. Veterinary Biological Product License or a U.S. Veterinary 

Biological Product Permit for antigen/antibody-based immunodiagnostic test kits for the 

diagnosis of animal disease and/or immunological status, as authorized by Title 9 Code 

of Federal Regulations (9 CFR), Part 114.9(f). In VS Memo 800.73, Part VII. Serial 

Release Testing C. Potency, guidance regarding the serial release potency requirements is 

provided. The purpose of this Center for Veterinary Biologics (CVB) Notice is to 

provide additional guidance for replacement of potency panel reference samples. 

VS Memo 800.73 states: “The manufacturer must perform a potency test on each serial 

of assembled test kits...as specified in the filed Outline of Production, using reference 

samples that are acceptable to APHIS. The purpose of the potency test is to ensure that 

each serial of the assay is producing accurate test results when properly performed. 

Serial release testing provides confidence that each serial will perform to the specificity 

and sensitivity standards determined at the time of licensure.” The 9 CFR Part 113.5, 

General Testing, states, “(a) No biological product shall be released prior to the 

completion of tests prescribed in a filed Outline of Production or Standard Requirement 

for the product to establish the product to be pure, safe, potent, and efficacious.” 

Performance characteristics for diagnostic test kits are determined during prelicense 

evaluation. The potency assay does not determine performance characteristics; it is 

intended to demonstrate that minimum potency standards are met throughout the dating 

of the product. 

VS Memo 800.73 further defines the panel of reference samples for potency testing of 

diagnostic test kits to include examples of negative/uninfected animals; strongly positive 

animals; weakly positive animals; samples generating assay values just above and just 

below the cutoff value between positive and negative classification; samples from 

 animals with reactivity to closely related (potentially cross-reactive) antigens and/or 

 vaccinated animals; and samples from animals reactive for only one, or a subset, of 

United States 

Department of 

Agriculture

Animal and Plant 

Health Inspection 

Service 

Veterinary Services 

Center for Veterinary 

Biologics 

510 S. 17th Street, 

Suite 104 

Ames, IA 50010 

(515) 232-5785 

FAX (515) 232-7120 
Center for Veterinary Biologics Notice No. 06-11 2 

antigens for kits that detect reactivity to more than one antigen. Although there is not a 

specific requirement that all samples are collected from individual animals, the CVB 

encourages firms to attempt to locate and characterize samples from individual animals 

that meet the target objectives. When it is not possible to locate appropriate samples, 

dilutions of positive samples may be approved in order to ensure availability of samples 

with desired reactivities. 

VS Memo 800.73 states, “The reference samples must be identified in the Outline of 

Production by lot number, date of preparation, purpose, and acceptable assay range. 

When the inventory of a reference sample is low, a replacement sample should be 

identified and validated. The replacement sample should serve the same purpose as the 

original sample (e.g., a strongly positive sample is replaced by another strongly positive 

sample). Validation data must be accepted by APHIS prior to use of the replacement 

sample in serial release testing; confirmatory testing at the CVB-L may be required.” 

To support replacement reference samples for potency panels, manufacturers should 

conduct an abbreviated validation study that aims to compare the reactivity of 

replacement and current reference samples. The CVB will evaluate the data based on the 

following criteria: 

1. The replacement reference sample should serve the same purpose as the sample it is 

replacing. Its performance should demonstrate its ability to fulfill the role of the 

sample it is intended to replace, but it need not have the precisely same reactivity 

(e.g., O.D. or S/P value) as that of the previously approved reference sample. 

2. The assessment should seek to illustrate the important characteristics of the reference 

samples’ reactivity, for example by estimating appropriate parameter estimates (e.g. 

mean and variance if the data are normally distributed) and/or graphical displays of 

the data distribution (e.g. scatter plots or box plots) for both new and current 

reference samples. The appropriate assessment of the validation data will vary by 

reference panel member. 

a. For reference samples with reactivity that is not near the extremes of the 

dynamic range (i.e. not near the diagnostic cutoff value, extinction, or 

saturation region), the following considerations will generally apply: 

i. For most reference panel members (e.g., positive samples not near the 

saturation or critical decision points of the assay), the replacement 

reference sample will be acceptable if the distributions of the current 

and replacement reference samples are similar. Minimum and 

maximum values for acceptable performance (reactivity range) should 

be established. If the current reference data are not available, 

reasonable judgment should be used to approve the acceptable 

replacement reference sample range of reactivity. 
Center for Veterinary Biologics Notice No. 06-11 3 

ii. Replacement low positive reference sample reactivity should be close 

to the critical decision point (cutoff value between positive and 

negative classification). For visual-read kits, the reactivity of the 

replacement reference sample throughout the dating period of the 

product should remain within the targeted response range (i.e., should 

remain positive). For kits in which instrumentation is used to 

determine reactivity (ELISAs or other kits read using instrumentation), 

reactivity ranges should be established with an appropriate response 

range near the cutoff value. Stability data may be helpful in predicting 

the appropriate target reactivity. 

b. For reference samples at the extremes of the dynamic range, such as negative 

reference samples, the reactivity of the replacement reference sample should 

be within the response range, (e.g., all negative reference samples should test 

within the negative range). 

i. For negative samples, the range should be stated as “equal to or less 

than (or more than, depending on the architecture of the assay) (the 

negative cut-off value).” 

ii. In general, samples that are so strongly positive that they test in, or 

near, the saturation region should not be used. If circumstances dictate 

that such samples are necessary, the low reactivity should be 

established and the reactivity range should be stated as “equal to or 

greater than (or less than, depending on the architecture of the assay) 

(the low reactivity).” 

3. Sufficient validation data should be developed to demonstrate the reference sample’s 

performance. The reference sample should be tested multiple times by multiple users, 

typically using multiple in-date serials of the licensed diagnostic test kit. One 

example of a generally acceptable protocol for replacing reference samples would 

include independent testing of the proposed reference sample by two or more 

competent technicians experienced in running the assay; using a minimum of two 

serials, each tested a minimum of five times; using the number of replicates of the 

replacement reference sample and current reference sample in each independent test 

as specified in the current filed Outline of Production. All serials used to develop the 

data must have satisfactory potency using the previously approved reference sample 

panel. We recommend that one of the serials used to develop the data should be near 

the release date and one of the serials should be near the expiration date. This will 

provide data to support the acceptable range of reactivity for the reference panel 

sample throughout product dating. The statistical, mathematical, or other method used 

to establish the proposed reactivity range should be stated. Reduced testing may be 

approved when sample quantities or serials are limited. 
Center for Veterinary Biologics Notice No. 06-11 4 

Prior to filing the Outlines of Production for approval of replacement reference samples, 

the CVB may require confirmatory testing at the CVB Laboratory to ensure that the 

replacement reference sample is appropriate and comparable to the previously approved 

reference sample. 

 /s/ Richard E. Hill 

Richard E. Hill, Jr. 

Director 

Center for Veterinary Biologics